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Degradation of the isoflavone biochanin a by isolates of Nectria haematococca (Fusarium solani)
Phytochemistry, Vol. 22, No. 7, pp. 1539-1541, 1983. Printed in Great Britain.
0031-9422/83 %3.00+0.00 PergamonPressLtd.
DEGRADATION OF THE ISOFLAVONE NECTRIA HAEMATOCOCCA U.
WILLEKE, K. M. WELTRING,
W.
BIOCHANIN A BY ISOLATES (FUSARIUM SOLANI) BARZ
and H. D.
OF
VANETTEN*
Lehrstuhl fiir Biochemie der Pflanzen, WestfSsche Wilhelm+Universitlt, D-4400 Miinster, West Germany; *Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. (Revised received 13 December 1982) Key Word Index--Nectria hnemntococca; Fusarium solani; isoflavonoids; biochanin A; pisatin; degradation; phytopathogenic fungi.
Abstract-Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for &jr virulence on pea plants and their &My to degrade the pbyIoa1exin p&tin were assayed for the catabotism o{ the isoffavone biochanin A (5,7-dihydrox@‘-methoxyisoffavone). Eleven isolates cataboIized the isoffavone along the pathway: biachanin A -+ dihydrobiochanm A + 3-(p-meChoxyp~enylj-6-Irydroxy-y-pyrone + p-methorcyphenylacetic acid + p-hydroxyphenylacetic acid ---t 3,4_dihydroxyphenylacetic acid.
INTRODUCTION
Higher plants possess a variety of defense mechanisms to prevent the penetration and the intracellular growth of ffun_ea!_narasites. Amow these mechanisms. the structurally diverse group of preinfectional inhibitors [l, 21, various other polyphenols [I] and the ever increasing number of phytoalexins [3&6] are considered to be important defense compounds. Several phytopathogenic fungi have recently been S%PJ%+Zi ‘RI US%% +L$&?&‘K~~J$0 A3idt I+& ACtt?k3fit
[ 111. In addition, one on the isolates (T-30) is insensitive to biochanin A and is pathogenic on chickpea Cicer arietinum [24], a plant that produces biochanin A. We ~K~~~~~~~~t~~~~~~~t~~ t&?&&&&!t~~~~ N .kX!Km&Qcocca a&o aiiFer ii~rtie atjiiliy rb atgrao’e tie preihr~cdbmn’ inhibitor biochanin A. Furthermore, the degradative pathway of 1 by N. haematococca and the sensitivity of this fungus to 1 were elucidated.
RESULTS
cvn~~~~~~~~~~RFfTJ';';~~.T~Rl~,lRf;"dl:"JLu~
palhogens can be considered 10 possess fne ability to degrade phytoaIexins 19, ID}, recent sInties with numerous isolates of Nectria haematococca, mating population VI (Fusarium so/&), have established an association between the ability to degrade the pea phytoalexin pisatin and virulence on peas [ 11, 12). All highly virulent strains were tolerant of pisatin and were able to detoxify the phytoalexin by 0-demethylation and degradation. In a series of other studies, numerous fungi of the genus Fusarium were shown to be potent degraders of both preinfectional inhibitors and phytoalexins with an isoflavonoid skeleton [7, 13-161. Thus, Fusarium jauanicum converted the isoflavone biochanin A (5,7-dihydroxy-4’methoxyisoflavone) (1) first to dihydrobiochanin A, which was subsequently degraded via 3-(p-methoxyphenyl)-6hydroxy-y-pyrone and p-hydroxyphenylacetic acid to 3,4dihydroxyphenylacetic acid [lS]. Other Fusarium species start biochanin A degradation by 4’-0-demethylation yielding genistein [ 171 or by 3’-hydroxylation [IS]. In this study, we have determined the ability of 12 isolates of N. haematococca to degrade the isoflavone biochanin A (1). This compound is occasionally considered to be a preinfectional inhibitor [19-211 and it was shown to occur in considerable concentrations in the rhizodermis of a variety of plants (e.g. Cicer arietinum, various Trrloliolium species, Baptisia australis and Ononis spinosa) of the Leguminosae [22, 231. The 12 selected isolates have previously been shown to differ in their ability to degrade pisation and in their virulence on pea
In three parallel seties 0F standard incubation assays with myceM preparatjons of Ihe 12 &dates of YI. haematococca [ll], the breakdown of biochanin A (1) (10e4 M) was folIowed for a period of up to 48 hr. AIiquots of the nutrient medium taken at 2 hr intervaIs were assayed for biochanin catabolites by TLC (S,, S,), UV-visible spectroscopy and spectroscopic means as described previously [15]. Thus, the chemical structures of the individual metabolites and the time period of their transient accumulation were determined. The structures of the degradation products are depicted in Fig. 1 where they are arranged in a sequence as indicated by the times of their maximum accumulation (Table 1). Except for isolate T-214 which seemed to lack the ability to degrade 1 all other isolates rapidly degraded the isoflavone. After a lag phase of some 2-3 hr, dihydrobiochanin A (2) was observed as the first conversion product with the pyrone, 3 being formed as the subsequent catabolite. Finally, phydroxyphenylacetic acid (5) and 3,4_dihydroxyphenylacetic acid (6) could be isolated from the nutrient medium. p-Methoxyphenylacetic acid (4) was detected in small amounts in three isolates only. Using [2-14C]biochanin A as substrate for several isolates the acids 5 and 6 could be isolated in labelled form which proves that the acetic acid side chain of 5 and 6 originates from carbon atoms 2 and 3 of biochanin A. Though 6 was not accumulated by the isolates T-8, T-30, T-78 and T-l 10, we assume that in these cases p-hydroxyphenylacetic acid is also catabolized via 3,4_dihydroxyphenylacetic acid.
1539
u.
1540
WILLEKE
Pt 01.
our studies rapidly degraded the preinfectional inhibitor biochanin A. The degradation of 1 again appeared to be substrate induced. Although the 11 isolates differ greatly in their ability to degrade the phytoalexin pisatin (Table I), there seems to be no significant qualitative or quantitative difference in the ability to degrade a 5-hydroxyisoflavone. These data suggest that the ability for phytoalexin degradation might be a more specific attribute than the ability to degrade a preinfectional inhibitor such as biochanin A. However, isolate T-214 apparently lacks the ability for the degradation of both
Fig. 1. Degradative pathway of biochanin A as carried out by isolates of Nectria haematococca in the sequence indicated by time periods of their maximum accumulation. Genistein (7) formation could not be detected with any isolate.
Genistein (7), which is the first degradation product of biochanin A with various other Fusarium species [13, 17, 253, could not be detected with any of the X. haematococca isolates used in these studies. The mycelial growth rate of all 12 isolates was essentially unaffected (7 to - 5 O0 inhibition) by biochanin A (1 x 1O-4 M). DISCUSSION
Eleven out of the 12 isolates of F. solani investigated Table
1. Catabolites
of blochanin
in
biochanin A and pisatin. The observation that this isolate was just as tolerant of biochanin A as were isolates that could degrade this compound was somewhat surprising. This could mean that degradation of biochanin A is not important for tolerance to biochanin A. or possibly degradation of biochanin A did not occur under the cultural conditions used for the metabolism studies but did under the cultural conditions used to bioassay T-214 for biochanin A sensitivity. Differences in cultural conditions have previously been shown to influence isoflavonoid degradation [ 11, 261. Although not all fungi of the genus Fusarium which have so far been investigated [7,13.17] possess the ability for isoflavone degradation. the great majority of the isolates of IV. haematococca (Table 1) are characterized by thik property. Furthermore, all the isolates used in this study that degraded biochanin A did so along the same pathway as shown in Fig. 1. This metabolic sequence has so far only been shown to occur with Fusurium jat~anicum [15]. while other F~tstlrium species either Odemethylate 1 to genistein (7). which is subsequently cleaved along different routes 113, 251, carry out 3’hydroxylation of 1 [lS]. O-methylate 1 in position 7 1181, or use other so far undescribed initial reactions of isoflavone catabolism [ 131. In general. studies of this type offer a means of investigating the great metabolic diversity of this group of fungi. Furthermore, the results corroborate that pathogenic strains of Fusurium species
A produced by isolates of Nrciria harmarococc~~ and the time period accumulation of these catabolites occurred. Catabolitea
Isolate*
Virulence on pea*
Degradation of pisatin”
Dihydrobiochanin A (2)
p-Hydroxyphenylacetic acid
3.4-Dihydroxyphenylacetic acid
(3)
(4)
(5)
(6)
n.d. n.d. n.d. n.d. n.d. n.d.
34 34 34 34 34 32
1l.d.
31
n.d. 34 n.d. 36 34 n.d. 34 n.d. 34
T-8
High
+
8-12
30
High High High Very Low Low Very Low Very Low Very
4-12 12-20 2- 6 4424 8 -12 12-m30 6 I2 12-24
12-30 2&30 12 -26 24-30 24-30 24 30 24-30 2430
low
+ + + _ + + _ + _
low
+ -
2-12 2-12
low
A
p-Methoxyphenylacetic acid
Phenylpyrone
T-27 T-30 T-63 T-69 T-78 T-86 T-110 T-200 T-214 T-224 T-231
low
of biochanin
(hr) when maximum
34 34 n.d. 34 No degradation of biochanin A observed 24-30 34 34 24-30 _2~__.~~~~~~~~~~~~~.~_~_
The time values represent the average of three determinations. *Isolate number and data on the virulence of these isolates on pea plants as well as their ability to degrade the phytoalexin from ref. [ll]. n.d., Catabolite not detected.
34 34
pisatin are
Fungal degradation of biochanin A possess the ability to metabolize those phenolic constituents of their host’s which are accumulated in the living plant tissue in very high concentrations 122, 231. EXPERIMENTAL Reagents.
Biochanin A, [2-‘%]biochanin A and reference samples of Biochanin A catabolites were from previous studies Cl51. Growthof fungi. Nectria haematococca isolates [11] were grown on Czapek-Dox medium and prepared for degradative studies according to earlier reports [15, 171. Standard incubation assays. Mycelia were collected by filtraticn, washed K 3 with KPi buffer (OX?5M; pH 7.5j and 3 g was inoculated into 100 ml of the same buffer containing biochanin A (10m4M). Substrate was predissolved in 0.5 ml 2-methoxyethanol prior to transfer into the buffer. These flasks were incubated at 30” and 160 rpm. Detection ofcatabolites. Samples (6 ml) of the standard incubation assays were &awn every 2 hr and mycelium ternwe& @ filtria’ilon. %er adi&%caiion :15X HJ%OA: _DX2-^, chrio&Aes were extracfeb &i~Et,D.The 0rparjlcSayer was recover&,&Q removed under vacuum and the residues were taken for TLC analysis as a methanolic soln. TLC was conducted on Si gel F2s4 plates with suitable reference compounds in CHC&Me(D)3 )3D:3) IS,> ana HD,%c-CHQ 1X7) j$) Catabolites were detected under a short wavelength UV-light and with spray reagents (fast blue salt B and diazotized paranitroaniline). The preparative isolation and the structural elucidations of catabolites by UV, MS and NMR techniques have previously been described [15]. RJs of the compounds are as folhows. In so1yen1S,: 1,0.5% 2,0.56; J,O.27; 4,0.4oP.5,0.>5; 6, 0.02. In solvent S,: 1,0.88; 2,0.91; 3, 0.65; 4,0.96; $0.6, 6,0.03.
Achnowledgernents-financia( support by the I%sts&e Forschungsgemeinschaft and Fonds der Chemischen Industrie (to W.B.) and by the Rockefeller Foundation grant No. RF79049*+W9. ($0H.D.Y.)js 13 a&A USD#tl’SEk $ZZ,,, I 5%23.56144-=+33-@ gratefully acknowledged. REFERENCES
1. Schanbeck, F. and Schliisser, E. (1976) in Physiological Plant
1541
P&&p (Heitefuss, R. and Williams, P. H., eds.) p. 653. Springer, Berlin. 2. Harborne, J. B. and Ingham, J. L. (1978) in Biochemical Aspects of Plant and Animalcoeuolution (Harborne, J. B., ed.) p. 343. Academic Press, New York. 3. Bailey, J. A. and Mansfield, J. W. (eds.) (1982) Phytoalexins. Blackie, Glasgow. KuC, J. A. (1976) in Physiological Plant Pathology (Heitefuss, R. and Williams, P. A., eds.) p. 632. Springer, Berlin. Ingham, J. L. (1979) Biochem. Syst. Ecof. 7, 29. Grisebach, H. and Ebel, J. (1978) Angew. Chem. 90,668. Barz, W., Willeke, U. and Weltring, K. M. (1980) Ann. Phytopathol. 12, 435. I. andSmith,D. A. \1980) Ann. Phytopathol. 12,453. 9. VanEtten, H. D., Matthews, D. E. and Smith, D. A. (1982) in Phytoalexins (Bailey, J. A. and Mansfield, J. W., eds.) p. 181.
a. KuYm,P.
Blackie, Glasgow. 10. Van den Heuvel, J. and Glazner, J. A. (1975) Ned. J. PIant Pathol.
81, 125.
&?TEiffl, If. D., IBPattlre%%, P. S., TegtmAw, X. I., D&u&, Ibr E. innr’ Sltiti, L I. ~l%X~At-j%alk!Rlmt’Rnthn! 24 2X? I.?. i%gmrefier,k J’.arId z%nErterr, K. 0. @&3j P~$,@%&7~ug~ 73, (in press). 13. Willeke, U. (1981) Doctoral thesis, University of Miinster, West Germany. 14 %?l-iir~ K.-M, Ban, w and X%&Z& 19 &i? (jP81) /I& Microbial. 130, 381.
15. Willeke, U. and Barz, W. (1982). Z. Naturjorsch. Teil C 37, 861.
16. Lappe, U. and Barz, W. (1978) Z. Naturjorsch. Teil C 33,301. 17. Barz, W., Schlepphorst, R. and Laimer, J. (1976) Phytoc&m&ry
IS, gZ K.-M., Mackenbrock, K. and Barz, W. (1982) Z. Naturforsch. Teil C 37, 570. 3. 0. (I%i) AclnCkewr. fenpr. 346, 23. 19 &e&&q,
18. Weltring,
20. Debnam, J. R. and Smith, 1. M. (19%) Physiol. Plant Pathol. *jr? 21. Kosuge, T. (1969) Annu. Reo. Pythopathol. 7, 195. 22. Gierse, H. D. (1975) Doctoral thesis, University of Freiburg/Br. 23. Kiister, J. (1982) Doctoral thesis, University of Miinster, West Germany. 24. Denny, T. P. and VanEtten, H. D. (1981) Physiol. Plant Pathol. 19, 419. 25. Willeke, U. and Barz, W. (1982) Arch. Microbial. 132, 266. 26. VanEtten, H. D.and Barz, W. (1981) Arch. Microbial. 129,56.
0031-9422/83 %3.00+0.00 PergamonPressLtd.
DEGRADATION OF THE ISOFLAVONE NECTRIA HAEMATOCOCCA U.
WILLEKE, K. M. WELTRING,
W.
BIOCHANIN A BY ISOLATES (FUSARIUM SOLANI) BARZ
and H. D.
OF
VANETTEN*
Lehrstuhl fiir Biochemie der Pflanzen, WestfSsche Wilhelm+Universitlt, D-4400 Miinster, West Germany; *Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. (Revised received 13 December 1982) Key Word Index--Nectria hnemntococca; Fusarium solani; isoflavonoids; biochanin A; pisatin; degradation; phytopathogenic fungi.
Abstract-Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for &jr virulence on pea plants and their &My to degrade the pbyIoa1exin p&tin were assayed for the catabotism o{ the isoffavone biochanin A (5,7-dihydrox@‘-methoxyisoffavone). Eleven isolates cataboIized the isoffavone along the pathway: biachanin A -+ dihydrobiochanm A + 3-(p-meChoxyp~enylj-6-Irydroxy-y-pyrone + p-methorcyphenylacetic acid + p-hydroxyphenylacetic acid ---t 3,4_dihydroxyphenylacetic acid.
INTRODUCTION
Higher plants possess a variety of defense mechanisms to prevent the penetration and the intracellular growth of ffun_ea!_narasites. Amow these mechanisms. the structurally diverse group of preinfectional inhibitors [l, 21, various other polyphenols [I] and the ever increasing number of phytoalexins [3&6] are considered to be important defense compounds. Several phytopathogenic fungi have recently been S%PJ%+Zi ‘RI US%% +L$&?&‘K~~J$0 A3idt I+& ACtt?k3fit
[ 111. In addition, one on the isolates (T-30) is insensitive to biochanin A and is pathogenic on chickpea Cicer arietinum [24], a plant that produces biochanin A. We ~K~~~~~~~~t~~~~~~~t~~ t&?&&&&!t~~~~ N .kX!Km&Qcocca a&o aiiFer ii~rtie atjiiliy rb atgrao’e tie preihr~cdbmn’ inhibitor biochanin A. Furthermore, the degradative pathway of 1 by N. haematococca and the sensitivity of this fungus to 1 were elucidated.
RESULTS
cvn~~~~~~~~~~RFfTJ';';~~.T~Rl~,lRf;"dl:"JLu~
palhogens can be considered 10 possess fne ability to degrade phytoaIexins 19, ID}, recent sInties with numerous isolates of Nectria haematococca, mating population VI (Fusarium so/&), have established an association between the ability to degrade the pea phytoalexin pisatin and virulence on peas [ 11, 12). All highly virulent strains were tolerant of pisatin and were able to detoxify the phytoalexin by 0-demethylation and degradation. In a series of other studies, numerous fungi of the genus Fusarium were shown to be potent degraders of both preinfectional inhibitors and phytoalexins with an isoflavonoid skeleton [7, 13-161. Thus, Fusarium jauanicum converted the isoflavone biochanin A (5,7-dihydroxy-4’methoxyisoflavone) (1) first to dihydrobiochanin A, which was subsequently degraded via 3-(p-methoxyphenyl)-6hydroxy-y-pyrone and p-hydroxyphenylacetic acid to 3,4dihydroxyphenylacetic acid [lS]. Other Fusarium species start biochanin A degradation by 4’-0-demethylation yielding genistein [ 171 or by 3’-hydroxylation [IS]. In this study, we have determined the ability of 12 isolates of N. haematococca to degrade the isoflavone biochanin A (1). This compound is occasionally considered to be a preinfectional inhibitor [19-211 and it was shown to occur in considerable concentrations in the rhizodermis of a variety of plants (e.g. Cicer arietinum, various Trrloliolium species, Baptisia australis and Ononis spinosa) of the Leguminosae [22, 231. The 12 selected isolates have previously been shown to differ in their ability to degrade pisation and in their virulence on pea
In three parallel seties 0F standard incubation assays with myceM preparatjons of Ihe 12 &dates of YI. haematococca [ll], the breakdown of biochanin A (1) (10e4 M) was folIowed for a period of up to 48 hr. AIiquots of the nutrient medium taken at 2 hr intervaIs were assayed for biochanin catabolites by TLC (S,, S,), UV-visible spectroscopy and spectroscopic means as described previously [15]. Thus, the chemical structures of the individual metabolites and the time period of their transient accumulation were determined. The structures of the degradation products are depicted in Fig. 1 where they are arranged in a sequence as indicated by the times of their maximum accumulation (Table 1). Except for isolate T-214 which seemed to lack the ability to degrade 1 all other isolates rapidly degraded the isoflavone. After a lag phase of some 2-3 hr, dihydrobiochanin A (2) was observed as the first conversion product with the pyrone, 3 being formed as the subsequent catabolite. Finally, phydroxyphenylacetic acid (5) and 3,4_dihydroxyphenylacetic acid (6) could be isolated from the nutrient medium. p-Methoxyphenylacetic acid (4) was detected in small amounts in three isolates only. Using [2-14C]biochanin A as substrate for several isolates the acids 5 and 6 could be isolated in labelled form which proves that the acetic acid side chain of 5 and 6 originates from carbon atoms 2 and 3 of biochanin A. Though 6 was not accumulated by the isolates T-8, T-30, T-78 and T-l 10, we assume that in these cases p-hydroxyphenylacetic acid is also catabolized via 3,4_dihydroxyphenylacetic acid.
1539
u.
1540
WILLEKE
Pt 01.
our studies rapidly degraded the preinfectional inhibitor biochanin A. The degradation of 1 again appeared to be substrate induced. Although the 11 isolates differ greatly in their ability to degrade the phytoalexin pisatin (Table I), there seems to be no significant qualitative or quantitative difference in the ability to degrade a 5-hydroxyisoflavone. These data suggest that the ability for phytoalexin degradation might be a more specific attribute than the ability to degrade a preinfectional inhibitor such as biochanin A. However, isolate T-214 apparently lacks the ability for the degradation of both
Fig. 1. Degradative pathway of biochanin A as carried out by isolates of Nectria haematococca in the sequence indicated by time periods of their maximum accumulation. Genistein (7) formation could not be detected with any isolate.
Genistein (7), which is the first degradation product of biochanin A with various other Fusarium species [13, 17, 253, could not be detected with any of the X. haematococca isolates used in these studies. The mycelial growth rate of all 12 isolates was essentially unaffected (7 to - 5 O0 inhibition) by biochanin A (1 x 1O-4 M). DISCUSSION
Eleven out of the 12 isolates of F. solani investigated Table
1. Catabolites
of blochanin
in
biochanin A and pisatin. The observation that this isolate was just as tolerant of biochanin A as were isolates that could degrade this compound was somewhat surprising. This could mean that degradation of biochanin A is not important for tolerance to biochanin A. or possibly degradation of biochanin A did not occur under the cultural conditions used for the metabolism studies but did under the cultural conditions used to bioassay T-214 for biochanin A sensitivity. Differences in cultural conditions have previously been shown to influence isoflavonoid degradation [ 11, 261. Although not all fungi of the genus Fusarium which have so far been investigated [7,13.17] possess the ability for isoflavone degradation. the great majority of the isolates of IV. haematococca (Table 1) are characterized by thik property. Furthermore, all the isolates used in this study that degraded biochanin A did so along the same pathway as shown in Fig. 1. This metabolic sequence has so far only been shown to occur with Fusurium jat~anicum [15]. while other F~tstlrium species either Odemethylate 1 to genistein (7). which is subsequently cleaved along different routes 113, 251, carry out 3’hydroxylation of 1 [lS]. O-methylate 1 in position 7 1181, or use other so far undescribed initial reactions of isoflavone catabolism [ 131. In general. studies of this type offer a means of investigating the great metabolic diversity of this group of fungi. Furthermore, the results corroborate that pathogenic strains of Fusurium species
A produced by isolates of Nrciria harmarococc~~ and the time period accumulation of these catabolites occurred. Catabolitea
Isolate*
Virulence on pea*
Degradation of pisatin”
Dihydrobiochanin A (2)
p-Hydroxyphenylacetic acid
3.4-Dihydroxyphenylacetic acid
(3)
(4)
(5)
(6)
n.d. n.d. n.d. n.d. n.d. n.d.
34 34 34 34 34 32
1l.d.
31
n.d. 34 n.d. 36 34 n.d. 34 n.d. 34
T-8
High
+
8-12
30
High High High Very Low Low Very Low Very Low Very
4-12 12-20 2- 6 4424 8 -12 12-m30 6 I2 12-24
12-30 2&30 12 -26 24-30 24-30 24 30 24-30 2430
low
+ + + _ + + _ + _
low
+ -
2-12 2-12
low
A
p-Methoxyphenylacetic acid
Phenylpyrone
T-27 T-30 T-63 T-69 T-78 T-86 T-110 T-200 T-214 T-224 T-231
low
of biochanin
(hr) when maximum
34 34 n.d. 34 No degradation of biochanin A observed 24-30 34 34 24-30 _2~__.~~~~~~~~~~~~~.~_~_
The time values represent the average of three determinations. *Isolate number and data on the virulence of these isolates on pea plants as well as their ability to degrade the phytoalexin from ref. [ll]. n.d., Catabolite not detected.
34 34
pisatin are
Fungal degradation of biochanin A possess the ability to metabolize those phenolic constituents of their host’s which are accumulated in the living plant tissue in very high concentrations 122, 231. EXPERIMENTAL Reagents.
Biochanin A, [2-‘%]biochanin A and reference samples of Biochanin A catabolites were from previous studies Cl51. Growthof fungi. Nectria haematococca isolates [11] were grown on Czapek-Dox medium and prepared for degradative studies according to earlier reports [15, 171. Standard incubation assays. Mycelia were collected by filtraticn, washed K 3 with KPi buffer (OX?5M; pH 7.5j and 3 g was inoculated into 100 ml of the same buffer containing biochanin A (10m4M). Substrate was predissolved in 0.5 ml 2-methoxyethanol prior to transfer into the buffer. These flasks were incubated at 30” and 160 rpm. Detection ofcatabolites. Samples (6 ml) of the standard incubation assays were &awn every 2 hr and mycelium ternwe& @ filtria’ilon. %er adi&%caiion :15X HJ%OA: _DX2-^, chrio&Aes were extracfeb &i~Et,D.The 0rparjlcSayer was recover&,&Q removed under vacuum and the residues were taken for TLC analysis as a methanolic soln. TLC was conducted on Si gel F2s4 plates with suitable reference compounds in CHC&Me(D)3 )3D:3) IS,> ana HD,%c-CHQ 1X7) j$) Catabolites were detected under a short wavelength UV-light and with spray reagents (fast blue salt B and diazotized paranitroaniline). The preparative isolation and the structural elucidations of catabolites by UV, MS and NMR techniques have previously been described [15]. RJs of the compounds are as folhows. In so1yen1S,: 1,0.5% 2,0.56; J,O.27; 4,0.4oP.5,0.>5; 6, 0.02. In solvent S,: 1,0.88; 2,0.91; 3, 0.65; 4,0.96; $0.6, 6,0.03.
Achnowledgernents-financia( support by the I%sts&e Forschungsgemeinschaft and Fonds der Chemischen Industrie (to W.B.) and by the Rockefeller Foundation grant No. RF79049*+W9. ($0H.D.Y.)js 13 a&A USD#tl’SEk $ZZ,,, I 5%23.56144-=+33-@ gratefully acknowledged. REFERENCES
1. Schanbeck, F. and Schliisser, E. (1976) in Physiological Plant
1541
P&&p (Heitefuss, R. and Williams, P. H., eds.) p. 653. Springer, Berlin. 2. Harborne, J. B. and Ingham, J. L. (1978) in Biochemical Aspects of Plant and Animalcoeuolution (Harborne, J. B., ed.) p. 343. Academic Press, New York. 3. Bailey, J. A. and Mansfield, J. W. (eds.) (1982) Phytoalexins. Blackie, Glasgow. KuC, J. A. (1976) in Physiological Plant Pathology (Heitefuss, R. and Williams, P. A., eds.) p. 632. Springer, Berlin. Ingham, J. L. (1979) Biochem. Syst. Ecof. 7, 29. Grisebach, H. and Ebel, J. (1978) Angew. Chem. 90,668. Barz, W., Willeke, U. and Weltring, K. M. (1980) Ann. Phytopathol. 12, 435. I. andSmith,D. A. \1980) Ann. Phytopathol. 12,453. 9. VanEtten, H. D., Matthews, D. E. and Smith, D. A. (1982) in Phytoalexins (Bailey, J. A. and Mansfield, J. W., eds.) p. 181.
a. KuYm,P.
Blackie, Glasgow. 10. Van den Heuvel, J. and Glazner, J. A. (1975) Ned. J. PIant Pathol.
81, 125.
&?TEiffl, If. D., IBPattlre%%, P. S., TegtmAw, X. I., D&u&, Ibr E. innr’ Sltiti, L I. ~l%X~At-j%alk!Rlmt’Rnthn! 24 2X? I.?. i%gmrefier,k J’.arId z%nErterr, K. 0. @&3j P~$,@%&7~ug~ 73, (in press). 13. Willeke, U. (1981) Doctoral thesis, University of Miinster, West Germany. 14 %?l-iir~ K.-M, Ban, w and X%&Z& 19 &i? (jP81) /I& Microbial. 130, 381.
15. Willeke, U. and Barz, W. (1982). Z. Naturjorsch. Teil C 37, 861.
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